The protein α-Synuclein (αS) is linked to Parkinson’s disease through its abnormal aggregation, which is thought to involve cytosolic and membrane-bound forms of αS. Following previous studies using micelles and vesicles, we present a comprehensive study of αS interaction with phospholipid bilayer nanodiscs. Using a combination of NMR-spectroscopic, biophysical, and computational methods, we structurally and kinetically characterize αS interaction with different membrane discs in a quantitative and site-resolved way. We obtain global and residue-specific αS membrane affinities, and determine modulations of αS membrane binding due to αS acetylation, membrane plasticity, lipid charge density, and accessible membrane surface area, as well as the consequences of the different binding modes for αS amyloid fibril formation. Our results establish a structural and kinetic link between the observed dissimilar binding modes and either aggregation-inhibiting properties, largely unperturbed aggregation, or accelerated aggregation due to membrane-assisted fibril nucleation.

Self-propagating β-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins. We here present an approach that utilizes dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of sparsely isotope-labeled proteins in frozen solution to take snapshots of the complete structural ensembles by exploiting the inhomogeneously broadened line-shapes. We investigated the intrinsically disordered protein α-synuclein (α-syn), which plays a key role in the etiology of Parkinson's disease, in three different physiologically relevant states. For the free monomer in frozen solution we could see that the so-called "random coil conformation" consists of α-helical and β-sheet-like conformations, and that secondary chemical shifts of neighboring amino acids tend to be correlated, indicative of frequent formation of secondary structure elements. Based on these results, we could estimate the number of disordered regions in fibrillar α-syn as well as in α-syn bound to membranes in different protein-to-lipid ratios. Our approach thus provides quantitative information on the propensity to sample transient secondary structures in different functional states. Molecular dynamics simulations rationalize the results.